Genistein is an isoflavone. Isoflavones are a subclass of flavonoids, natural products typically isolated in glycosylated form from plants. The aglycone is the biologically active form that has the most medical and commercial interest.
The glycosylated form of genistein is known as genistin. The preparation of genistein must include a step in which the core isoflavone structure (genistein) is separated from the glucose moiety. Genistin, which is found in soybeans, is convened to the biologically active form, genistein, through the action of a beta-glucosidase enzyme.
Demand for genistein is expected to increase significantly in the near future. Genistein is becoming an established research tool as an inhibitor of protein tyrosine kinase, an enzyme involved in increased cell proliferation. Protein tyrosine kinase (PTK), is one of genistein's well studied biological targets. PTK is known to give cells a proliferative advantage. A variety of oncogenes of the Src family have been shown to have tyrosine kinase activity. Genistein is a PTK inhibitor. Genistein inhibited the PTK activities of EGF-receptor and pp60v-src with an ID50 of 6 and 7 .mu.g/ml respectively in experiments using purified components, but in intact A431 cells 40 .mu.g/ml were required to inhibit the cellular phosphotyrosine levels. Genistein is reported to be an inhibitor of eukaryotic DNA topoisomerase (topo) I and II.
Genistein is used as a chemopreventive agent in animal studies, is required for clinical trials as a dietary supplement; and likely has application as a chemotherapeutic agent when coupled to anti-tumor specific antibodies.
Populations consuming soybeans show reduced incidence of breast, colon, and prostate cancer. Initial animal studies suggested that two isoflavones, genistein and daidzein, may be the active ingredients of soybeans that function as chemopreventive agents. Genistein injected into neonatal rats reduces dimethylbenz(a)anthracene (DMBA)-induced mammary tumor induction by about 50%. Either genistein or daidzein provides some protection against N-methyl N-nitrosurea (MNU)-induced mammary tumors in rats. Although genistein was effective in reducing both the tumor incidence and multiplicity, daidzein reduced only multiplicity in the later studies.
Currently genistein is being tested by the National Cancer Institute against colon carcinogenesis, as part of the Chemoprevention Screening Program, and early results show effectiveness against colon papillomas. Clinical trials to identify the metabolism and pharmacokinetics of genistein (phase I) are also being currently performed.
A recent study identified genistein as a potent antitumor agent (when coupled with antitumor specific antibodies) against colon carcinogenesis of human B-cell leukemia in immunodeficient mice. The B43-genistein immunoconjugate at less than one-tenth the maximum tolerated dose killed more than 99% of human leukemia cells. The postulated mechanism of genistein's action in this study was that of PTK inhibitor.
Because of the potential clinical use of genistein as a chemopreventive and/or chemotherapeutic agent it is becoming essential to produce this isoflavone and some of its promising structural analogs in larger quantities and to reduce its price. Genistein is currently expensive to produce using current technology. Chemically synthesized genistein or genistein extracted from soybeans currently can cost up to $5,000 a gram. However, the compound is not produced routinely in kilogram quantities at present.
The present invention provides an inexpensive process for the large scale production of genistein wherein Saccharopolyspora erythraea, the organism that is used in the commercial production of erythromycin, is fermented on a soy-based fermentation medium.